Methods for the manufacture of liposomal drug formulations

ABSTRACT

Provided herein is a method for the large-scale manufacture of liposomal drug formulations containing an aminoglycoside such as amikacin having advantageous lipid/drag characteristics. The method utilizes a particular relative flow rate ratio of lipid to drug streams to obtain liposomes with a high aminoglycoside encapsulation efficiency. The resulting liposomal drug formulations advantageously comprise an overall lipid-to-drug weight ratio of less than 1:1.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional Application Ser.No. 62/665,564, filed May 2, 2018, the disclosure of which isincorporated by reference herein in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Liposomal drug formulations enable the ability to target and enhance theuptake of active agents at specific sites of disease. Such formulationshave been developed to treat various pulmonary disorders, includingthose caused by pulmonary infections, where their characteristics makethem an ideal choice for the inhalation delivery of anti-infectiveagents.

One such anti-infective agent, amikacin, has been packaged in liposomes,and has been studied in multiple clinical trials in adult patients forthe treatment of refractory nontuberculous mycobacterial (NTM) lungdisease cause by Mycobacterium avium complex (MAC). In a recent Phase 3study of the amikacin liposome inhalation suspension (ALIS), it wasdemonstrated that the addition of ALIS to guideline based therapy (GBT)eliminated evidence of NTM lung disease caused by MAC in sputum by month6 in 29% of patients, compared to 9% of patients on GBT alone.

Although liposomes containing a relatively high amikacin to lipid ratiohave been prepared at the bench scale, it is well-known that it is not aroutine matter to scale up such processes to produce, at the commercialmanufacturing scale, liposomal formulations where parameters such asdrug concentration, amount of lipid in the formulation, lipid-to-drugratio, captured volume, drug leakage, viscosity, and particle size areconsistently maintained within specification for clinical and/orcommercial use.

The present invention addresses the need for a repeatable large-scaleprocess for preparing liposomes containing an aminoglycoside antibioticsuch as amikacin, and having a high aminoglycoside-to-lipid weight ratio(and in turn, a low lipid-to-aminoglycoside weight ratio) and superiorencapsulation efficiency.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for thelarge-scale manufacture of a liposomal aminoglycoside formulationcomprising a lipid and an aminoglycoside (e.g., amikacin), with a highaminoglycoside loading relative to the lipid concentration (i.e., a highrelative weight ratio of aminoglycoside-to-lipid). In particular, thelipid-to-aminoglycoside (e.g., amikacin) weight ratio (also referred toas the “L/D weight ratio”) in the liposomal suspension preparedaccording to method of the present invention is less than 1:1 uponcompletion of the process, for example between about 0.5:1 and about0.9:1. In one embodiment, the lipid-to-aminoglycoside weight ratio ofthe liposomal suspension prepared according to the method of the presentinvention is about 0.7:1 (lipid:aminoglycoside) upon completion of themanufacturing process.

In another aspect, the present invention relates to a method for thelarge-scale manufacture of a liposomal drug formulation comprising alipid and aminoglycoside (e.g., amikacin), wherein the aminoglycoside iscontained within the liposome with a high encapsulation efficiency(e.g., an encapsulation efficiency of at least about 40% prior towashing to remove free aminoglycoside from the formulation).

In one embodiment, the method comprises mixing a first stream comprisinga lipid with a second stream comprising an aminoglycoside to form acombined lipid-aminoglycoside stream. The combined lipid-aminoglycosidestream comprises liposomally encapsulated aminoglycoside, which in oneembodiment, is formed at the intersection of the lipid stream and theaminoglycoside stream. In a further embodiment, the lipid-aminoglycosidestream is mixed with an aqueous saline solution in a reaction vessel(see, e.g., FIG. 1). In one embodiment, the aminoglycoside is present inan aqueous solution prior to the mixing step. In another embodiment, thelipid is present in an alcoholic solution, e.g., an ethanolic solution,prior to the mixing step. In a further embodiment, the lipid comprises aphospholipid and cholesterol. In one embodiment, the relative flow rateratio of the second stream comprising aminoglycoside to the first streamcomprising a lipid is about 1.5:1 (aminoglycoside stream:lipid stream)to about 2:1 (aminoglycoside stream:lipid stream). In a furtherembodiment, the lipid comprises dipalmitoylphosphatidylcholine (DPPC)and cholesterol.

In one embodiment, the aqueous saline solution is added to the reactionvessel via a third stream. In a further embodiment, the third stream isadded to the reaction vessel at the same time as the combinedlipid-aminoglycoside stream. In another embodiment, the third stream isadded to the reaction vessel prior to the addition of the combinedlipid-aminoglycoside stream to the reaction vessel. In anotherembodiment, the aqueous saline solution is at about room temperatureprior to entering the reaction vessel. In one embodiment, the aqueoussaline solution is about 1.5% aqueous sodium chloride.

In one particular aspect, the present invention provides a method forthe large-scale manufacture of a liposomal drug formulation comprising alipid and aminoglycoside (e.g., amikacin), wherein the aminoglycoside isencapsulated within or complexed with the liposome, prior to a washingstep, at an encapsulation efficiency of at least 40%. Following thewashing step, which in one embodiment, is carried out via tangentialflow filtration, the weight ratio of lipid-to-aminoglycoside in theliposomal aminoglycoside formulation is less than 1:1, for examplebetween about 0.5:1 and about 0.9:1 (e.g., about 0.7:1). In oneembodiment of this method, the aminoglycoside is amikacin. In a furtherembodiment, the amikacin is present as amikacin sulfate.

In one embodiment, a first stream comprising a lipid is mixed with asecond stream comprising an aminoglycoside to form a combinedlipid-aminoglycoside stream (e.g., a lipid-amikacin stream) comprisingliposomal aminoglycoside. In one embodiment, the liposomalaminoglycoside formulation is formed at the intersection of the twostreams, i.e., upon formation of the combined lipid-aminoglycosidestream. In a further embodiment, the flow rate of the first streamcomprising a lipid is from about 0.5 kg/min to about 1.5 kg/min and theflow rate of the second stream comprising aminoglycoside is from about 1kg/min to about 2 kg/min. In a further embodiment, the flow rate of thefirst stream comprising a lipid is from about 3 kg/min to about 4 kg/minand the flow rate of the second stream comprising the aminoglycoside isfrom about 5 kg/min to about 7 kg/min. In another embodiment, therelative flow rate ratio of the second stream comprising aminoglycosideto the first stream comprising a lipid is about 1.5:1 (aminoglycosidestream flow rate:lipid stream flow rate) to about 2:1 (aminoglycosidestream flow rate:lipid stream flow rate). In yet another embodiment, thelipid comprises dipalmitoylphosphatidylcholine (DPPC) and cholesterol.

In one embodiment, the method for the large-scale manufacture of aliposomal drug formulation comprises mixing a first stream comprising alipid with a second stream comprising aminoglycoside to form a combinedlipid-aminoglycoside stream, and adding the combinedlipid-aminoglycoside stream to a vessel comprising an aqueous salinesolution. The aqueous saline solution, in one embodiment, is added tothe reaction vessel via a third stream (see, e.g., FIG. 1).

In a further embodiment, when the flow rate of the first streamcomprising a lipid is from about 0.5 kg/min to about 1.5 kg/min and theflow rate of the second stream comprising aminoglycoside is from about 1kg/min to about 2 kg/min, the flow rate of the third stream is fromabout 0.5 L/min and about 2.0 L/min, for example, from about 1.0 L/minto about 2.0 L/min, e.g. from about 1.0 L/min to about 1.5 L/min,including about 1.25 L/min. In another embodiment, when the flow rate ofthe first stream comprising a lipid is from about 3 kg/min to about 4kg/min and the flow rate of the second stream comprising aminoglycosideis from about 5 kg/min to about 7 kg/min, the flow rate of the thirdstream is from about 3 L/min and about 6 L/min, for example, from about4 L/min to about 6 L/min, e.g. from about 4.5 L/min to about 5.5 L/min,including about 5 L/min.

As used herein, except where specifically stated otherwise, the term“aminoglycoside” is intended to include the aminoglycoside free base andany pharmaceutically acceptable salt thereof. For example, the term“amikacin” is intended to include amikacin free base and anypharmaceutically acceptable salt thereof (e.g., amikacin sulfate).

In one embodiment, the method for the large-scale manufacture of aliposomal aminoglycoside (e.g. amikacin) formulation comprises mixing afirst stream comprising a lipid comprising a phospholipid with a secondstream comprising aminoglycoside (e.g. amikacin) to form a combinedlipid-aminoglycoside stream. In a further embodiment, thelipid-aminoglycoside stream is mixed with an aqueous saline solution ina reaction vessel. In one embodiment, the phospholipid is aphosphatidylcholine. Ina further embodiment, the phosphatidylcholine isDPPC. In another embodiment, the lipid comprises a phospholipid and asterol. In a further embodiment, the sterol is cholesterol. In oneembodiment, the lipid comprises DPPC and cholesterol.

In one embodiment, the method for the large-scale manufacture of aliposomal aminoglycoside formulation comprises mixing a first streamcomprising a lipid with a second stream comprising aminoglycoside,wherein the first stream is mixed with the second stream to form acombined lipid-aminoglycoside stream. The combined lipid-aminoglycosidestream comprises liposomal aminoglycoside. The liposomal aminoglycosidein one embodiment, is formed upon mixing the first stream and secondstream, e.g., at the intersection of the two streams. In a furtherembodiment, the combined lipid-aminoglycoside stream is added to areaction vessel and mixed with an aqueous saline solution. In a furtherembodiment, the aminoglycoside stream and the lipid stream are eachmaintained at a temperature from about 30° C. to about 50° C. prior tomixing. In a further embodiment, the aminoglycoside and lipid streamsare each maintained at a temperature of from about 35° C. to about 45°C., for example from about 38° C. to about 42° C. prior to mixing. Inone embodiment, the combined lipid-aminoglycoside stream is cooled uponentering the reaction vessel. In another embodiment, the combinedlipid-aminoglycoside stream is cooled by the aqueous saline solution inthe reaction vessel. In one embodiment, the reaction vessel ismaintained at a temperature from about 25° C. and about 40° C., e.g.,from about 27° C. to about 35° C. In another embodiment, the reactionvessel is maintained at a temperature of about 30° C. In anotherembodiment, the reaction vessel is maintained at a temperature of about33° C.

In another aspect of the invention, a liposomal aminoglycosideformulation is manufactured on a large-scale according to a methodprovided herein. In one embodiment, the concentration of aminoglycoside(e.g. amikacin) present in the liposomal drug formulation so prepared isabout 10 g/L or greater, for example from about 50 g/L to about 100 g/L,including about 60 g/L to about 80 g/L and about 65 g/L to about 75 g/L(e.g., about 20 g/L, about 30 g/L, 40 about g/L, about 50 g/L, about 60g/L, about 70 g/L or about 80 g/L). In a further embodiment, theconcentration of lipid present in the liposomal drug formulation soprepared is from about 10 g/L to about 100 g/L, including about 20 g/Lto about 80 g/L and about 40 g/L to about 60 g/L (e.g. about 50 g/L). Inanother embodiment, the L/D ratio of a liposomal drug formulationmanufactured on a large-scale according to a method provided herein isless than 1:1, for example between about 0.5:1 and about 0.8:1 (e.g.about 0.7:1).

In another embodiment, the liposomal drug formulation manufactured on alarge-scale according to a method provided herein comprises liposomeparticles with a mean particle size (i.e. a mean diameter) of from about200 nm to about 500 nm, for example from about 200 nm to about 400 nm(e.g. from about 250 nm to about 350 nm).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicting one embodiment of the invention for preparing aliposomal aminoglycoside formulation.

FIG. 2 shows the effect of relative lipid/amikacin flow rates on theresulting L/D ratio of various liposomal amikacin formulations.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention described herein relates to a method formanufacturing a liposomal aminoglycoside formulation on a large-scale.In one embodiment, the method comprises mixing a first stream comprisinga lipid (also referred to herein as a “lipid stream”) with a secondstream comprising an aminoglycoside such as amikacin (also referred toherein as a “drug stream”) to form a combined lipid-aminoglycosidestream, and the lipid-aminoglycoside stream is mixed with an aqueoussaline solution in a reaction vessel. In some embodiments, the aqueoussaline solution enters the reaction vessel via a third stream.

The mixing of the lipid and drug streams is effected such that aturbulent flow results when forming the combined lipid-aminoglycosidestream. A turbulent flow is conveniently achieved using an appropriateT-shaped or Y-shaped infusion module for “in-line” mixing of the lipidand drug streams.

The term “large-scale” means the use of at least about 5 kgaminoglycoside base starting material in the drug stream (calculated toat least about 5 kg aminoglycoside base if a pharmaceutically acceptablesalt is used). In one embodiment, about 5 kg to about 50 kgaminoglycoside base starting material is used, for example about 5 kg toabout 35 kg aminoglycoside base starting material. In one embodiment, atleast about 8 kg aminoglycoside base starting material is used. Inanother embodiment, at least about 30 kg aminoglycoside base startingmaterial is used. In one embodiment, the aminoglycoside is amikacin(e.g. amikacin sulfate).

The aminoglycoside used in the methods provided herein can be present asa pharmaceutically acceptable salt or as the free base. As providedabove, in one embodiment, the aminoglycoside is amikacin, e.g., amikacinsulfate.

In another embodiment, the aminoglycoside is amikacin, apramycin,arbekacin, astromicin, capreomycin, dibekacin, framycetin, gentamicin,hygromycin B, isepamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodestreptomycin, ribostamycin, sisomicin, spectinomycin, streptomycin,tobramycin, verdamicin, or a combination thereof.

In yet another embodiment, the aminoglycoside is AC4437, amikacin,apramycin, arbekacin, astromicin, bekanamycin, boholmycin, brulamycin,capreomycin, dibekacin, dactimicin, etimicin, framycetin, gentamicin,H107, hygromycin, hygromycin B, inosamycin, K-4619, isepamicin, KA-5685,kanamycin, neomycin, netilmicin, paromomycm, plazomicin, ribostamycin,sisomicm, rhodestreptomycin, sorbistin, spectinomycin, sporaricin,streptomycin, tobramycin, verdamicin, vertilmicin, or a combinationthereof.

A “pharmaceutically acceptable salt” includes both acid and baseaddition salts. A pharmaceutically acceptable addition salt refers tothose salts which retain the biological effectiveness and properties ofthe free bases, which are not biologically or otherwise undesirable, andwhich are formed with inorganic acids such as, but are not limited to,hydrochloric acid (HCl), hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid (e.g., as lactate), lactobionic acid,lauric acid, maleic acid, malic acid, malonic acid, mandelic acid,methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, aceticacid (e.g., as acetate), tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid (TFA), undecylenic acid,and the like. In one embodiment, the pharmaceutically acceptable salt isHCl, TFA, lactate or acetate. In one embodiment, the pharmaceuticallyacceptable salt is a sulfate salt, e.g., amikacin sulfate.

“Liposomal aminoglycoside formulation” refers to a lipid-aminoglycosideformulation wherein the lipid is in the form of a liposome and theaminoglycoside is encapsulated by the liposome bilayer, or complexedwith the liposome bilayer. Liposomes are completely closed lipid bilayermembranes containing an entrapped aqueous volume. Liposomes may beunilamellar vesicles (possessing a single membrane bilayer) ormultilamellar vesicles (onion-like structures characterized by multiplemembrane bilayers, each separated from the next by an aqueous layer) ora combination thereof. The bilayer is composed of two lipid monolayershaving a hydrophobic “tail” region and a hydrophilic “head” region. Thestructure of the membrane bilayer is such that the hydrophobic(nonpolar) “tails” of the lipid monolayers orient toward the center ofthe bilayer while the hydrophilic “heads” orient towards the aqueousphase.

In one embodiment, the lipid-aminoglycoside formulation is manufacturedvia a method comprising a two-stream infusion process. In oneembodiment, the method comprises mixing a first lipid stream with asecond aminoglycoside stream in a T-shaped infusion module or Y-shapedinfusion module. The terms “T-shaped infusion module,” and “Y-shapedinfusion module” as used herein, refer to a T-shaped or Y-shaped chamberin which two or more streams are combined, for example, in which a lipidstream and a drug stream are combined to form a singlelipid-aminoglycoside stream. See, e.g., the diagram at FIG. 1. It willbe appreciated that the infusion module will have a bore sizeappropriate for the required rate of the lipid and drug streams used.Examples of suitable bore sizes include, but are not limited to, 3/16″and ⅜″.

In one embodiment, the first stream (lipid stream) comprises analcoholic (e.g., ethanolic) lipid solution. In one embodiment, thesecond stream (aminoglycoside stream) comprises an aqueousaminoglycoside solution (e.g., aqueous amikacin solution). In oneembodiment, the first and second streams are mixed to form a combinedlipid-aminoglycoside stream. In one embodiment, the first and secondstreams each enter the infusion module and the first and second streamsare mixed in the infusion module. In a further embodiment, the combinedlipid-aminoglycoside stream exits the infusion module and subsequentlyenters the reaction vessel. See FIG. 1.

In one embodiment, the combined lipid-aminoglycoside stream is mixedwith an aqueous saline solution in a reaction vessel, e.g., the samereaction vessel that the combined lipid-aminoglycoside stream entersafter exiting the infusion module. In one embodiment, the aqueous salinesolution comprises about 0.5-2% aqueous sodium chloride solution (e.g.,about 1.5%). In one embodiment, the saline solution is added to thereaction vessel prior to the combined lipid-aminoglycoside stream. Inanother embodiment, the saline solution is added to the reaction vesselat or about the same time as the combined lipid-aminoglycoside stream.In a further embodiment, the saline solution is added to the reactionvessel via a third stream. Thus, in some embodiments, thelipid-aminoglycoside formulation is manufactured via a method comprisinga 3-stream infusion process. In some embodiments, the third stream isadded to the reaction vessel separately from the combinedlipid-aminoglycoside stream.

In one embodiment, the combined lipid-aminoglycoside stream comprisesliposomal aminoglycoside, (e.g. amikacin), wherein the encapsulationefficiency of the aminoglycoside within the liposomes (or complexed tothe liposomes) is at least about 40%. “Encapsulation efficiency”, asused herein, refers to the amount of aminoglycoside encapsulated orcomplexed with liposomes prior to a filtration step, e.g., tangentialflow filtration of the liposomal aminoglycoside formulation to removefree aminoglycoside. For example, an encapsulation efficiency of betweenabout 400% and about 70% (e.g., from about 45% to about 55%) can beachieved by mixing the lipid and aminoglycoside (e.g. amikacin) streamsaccording to the method of this invention as herein described.

In one embodiment, the aminoglycoside stream and the lipid stream areeach maintained at a temperature from about 30° C. to about 50° C. priorto mixing the two streams. In a further embodiment, the aminoglycosideand lipid streams are each maintained at a temperature of from about 35°C. to about 45° C., for example from about 38° C. to about 42° C. priorto mixing. In another embodiment, the combination of the lipid andaminoglycoside solutions exhibits exothermal behavior. The temperatureof the combined lipid-aminoglycoside stream, in one embodiment, is fromabout 40° C. to 55° C. In a further embodiment, the temperature of thecombined lipid-aminoglycoside stream is from about 45° C. to about 50°C. In another embodiment, the combined lipid-aminoglycoside stream ismixed with an aqueous saline solution in a reaction vessel, wherein theaqueous saline solution is maintained at room temperature prior tomixing with the combined lipid-aminoglycoside stream. In anotherembodiment, an aqueous saline solution is added to the reaction vesselvia a third stream, wherein the third stream is maintained at roomtemperature prior to mixing with the combined lipid-aminoglycosidestream. In one embodiment, the combined lipid-aminoglycoside stream iscooled upon entering the reaction vessel. In another embodiment, thecombined lipid-aminoglycoside stream is cooled by the aqueous salinesolution in the reaction vessel. In another embodiment, the combinedlipid-aminoglycoside stream is cooled upon entering the reaction vessel.In one embodiment, the reaction vessel is maintained at a temperaturefrom about 25° C. and about 40° C., e.g., from about 27° C. to about 35°C. In another embodiment, the reaction vessel is maintained at atemperature of about 30° C. In another embodiment, the reaction vesselis maintained at a temperature of about 33° C.

In one embodiment, the lipid component of the liposomal drug formulationmanufactured by the method provided herein comprises electrically netneutral lipids, positively charged lipids, negatively charged lipids, ora combination thereof. In another embodiment, the lipid componentcomprises electrically net neutral lipids. In a further embodiment, thelipid component consists essentially of electrically net neutral lipids.In even a further embodiment, the lipid is DPPC and cholesterol.

The lipids used in the manufacture of the liposomal formulations of thepresent invention can be synthetic, semi-synthetic ornaturally-occurring lipids, including one or more of phospholipids,tocopherols, sterols, fatty acids, negatively-charged lipids andcationic lipids. In one embodiment, the lipid component consists ofelectrically neutral lipids, e.g., a sterol and a phospholipid.

In one embodiment, at least one phospholipid is present in the liposomaldrug formulation. In one embodiment, the phospholipid isphosphatidylcholine (PC), phosphatidylglycerol (PG),phosphatidylinositol (PI), phosphatidylserine (PS),phosphatidylethanolamine (PE), phosphatidic acid (PA), soyphosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soyphosphatidylserine (SPS), soy phosphatidylinositol (SPI), soyphosphatidylethanolamine (SPE), and soy phosphatidic acid (SPA);hydrogenated egg and soya counterparts (e.g., hydrogenated eggphosphatidylcholine and hydrogenated soy phosphatidylcholine),phospholipids made up of ester linkages of fatty acids in the 2 and 3 ofglycerol positions containing chains of 12 to 26 carbon atoms anddifferent head groups in the 1 position of glycerol that includecholine, glycerol, inositol, serine, ethanolamine, as well as thecorresponding phosphatidic acids. The carbon chains on these fatty acidscan be saturated or unsaturated, and the phospholipid may be made up offatty acids of different chain lengths and different degrees ofunsaturation.

In one embodiment, the lipid component of the liposomal drug formulationmanufactured by the method provided herein comprises aphosphatidylcholine. For example, in one embodiment, the lipid componentin the liposomal drug formulation comprisesdipalmitoylphosphatidylcholine (DPPC). In one embodiment, the lipidcomponent of the liposomal drug formulation comprises DPPC and a sterol,for example DPPC and cholesterol. Alternatively, the lipid consistsessentially of DPPC and cholesterol, or consists of DPPC andcholesterol. In a further embodiment, the DPPC and cholesterol have amolar ratio in the range of from about 19:1 (DPPC:cholesterol) to about1:1 (DPPC:cholesterol), or from about 9:1 (DPPC:cholesterol) to about1:1 (DPPC:cholesterol), or from about 4:1 (DPPC:cholesterol) to about1:1 (DPPC:cholesterol), or from about 2:1 (DPPC:cholesterol) to about1:1 (DPPC:cholesterol). In even a further embodiment, the DPPC andcholesterol have a molar ratio of about 2:1 (DPPC:cholesterol).

Other examples of lipid components of the liposomal drug formulationmanufactured by the method provided herein include, but are not limitedto, dimyristoylphosphatidycholine (DMPC),dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidcholine(DPPC), dipalmitoylphosphatidylglycerol (DPPG),distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol(DSPG), dioleylphosphatidyl-ethanolamine (DOPE), mixed phospholipidssuch as palmitoylstearoylphosphaidyl-choline (PSPC), and single acylatedphospholipids, for example, mono-oleoyl-phosphatidylethanolamine (MOPE).

Examples of sterol compounds in the liposomal drug formulationmanufactured by the method provided herein include, but are not limitedto, cholesterol, esters of cholesterol including cholesterolhemi-succinate, salts of cholesterol including cholesterol hydrogensulfate and cholesterol sulfate, ergosterol, esters of ergosterolincluding ergosterol hemi-succinate, salts of ergosterol includingergosterol hydrogen sulfate and ergosterol sulfate, lanosterol, estersof lanosterol including lanosterol hemi-succinate, salts of lanosterolincluding lanosterol hydrogen sulfate, lanosterol sulfate andtocopherols. The tocopherols include tocopherols, esters of tocopherolsincluding tocopherol hemi-succinates, salts of tocopherols includingtocopherol hydrogen sulfates and tocopherol sulfates. The term “sterolcompound” includes sterols, tocopherols and the like. Tocopherols andtheir water-soluble derivatives have been used to form liposomes, see,e.g., PCT Publication No. 87/02219.

In one embodiment, the concentration of lipid in the first stream isfrom about 10 g/L to about 50 g/L, or from about 10 g/L to about 30 g/L,or from about 15 g/L to about 25 g/L. In one embodiment, theconcentration of lipid in the first stream is about 17 g/L, about 18g/L, about 19 g/L, about 20 g/L, about 21 g/L, about 22 g/L, about 23g/L, about 24 g/L or about 25 g/L. In one embodiment, the concentrationof lipid in the first stream is about 20 g/L

In one embodiment, the concentration of aminoglycoside in the secondstream (aminoglycoside stream) is from about 10 g/L to about 100 g/L; orfrom about 20 g/L to about 70 g/L; or from about 30 g/L to about 60 g/L;or from about 40 g/L to about 50 g/L. In one embodiment, theconcentration of drug in the second stream is about 4 g/L, about 42 g/L,about 43 g/L, about 44 g/L, about 45 g/L, about 46 g/L, about 47 g/L,about 48 g/L, about 49 g/L or about 50 g/L. In one embodiment, theconcentration of aminoglycoside in the second stream is about 45 g/L. Ina further embodiment, the aminoglycoside is amikacin.

In one embodiment of the invention, the pH of the aminoglycoside streamis from 6 to about 7, or from about 6.5 to about 7.0. In a furtherembodiment, the pH of the aminoglycoside stream is about 6.7. Theaminoglycoside stream pH may be adjusted to the appropriate pH using asuitable base, such as an alkali or alkaline earth metal hydroxide, e.g.sodium hydroxide.

In another embodiment, the aqueous saline solution comprises about 0.5%sodium chloride to about 3% sodium chloride, for example about 0.75%,about 1.0%, about 1.25%, about 1.5%, about 1.75%, about 2.0%, or about2.5% sodium chloride. In one embodiment, the aqueous saline solutioncomprises about 1.5% sodium chloride.

In one embodiment, the flow rate of the lipid stream is from about 0.5kg/min to about 1.5 kg/min and the flow rate of the aminoglycosidestream is from about 1 kg/min to about 2 kg/min.

In a further embodiment, the flow rate of the lipid stream is from about3 kg/min to about 4 kg/min and the flow rate of the drug stream is fromabout 5 kg/min to about 7 kg/min. In another embodiment, the relativeflow rate ratio of the aminoglycoside stream to the lipid stream isabout 1.5:1 (aminoglycoside stream flow rate:lipid stream flow rate) toabout 2:1 (aminoglycoside stream flow rate:lipid stream flow rate).

In one embodiment, when the flow rate of the lipid stream is from about0.5 kg/min to about 1.5 kg/min and the flow rate of the aminoglycosidestream is from about 1 kg/min to about 2 kg/min, the flow rate of thethird stream comprising aqueous saline solution is from about 0.5 L/minand about 2.0 L/min, for example, from about 1.0 L/min to about 2.0L/min, e.g., from about 1.0 L/min to about 1.5 L/min, including about1.25 L/min. In another embodiment, when the flow rate of the lipidstream is from about 3 kg/min to about 4 kg/min and the flow rate of theaminoglycoside stream is from about 5 kg/min to about 7 kg/min, the flowrate of the third stream comprising the aqueous saline solution is fromabout 3 L/min and about 6 L/min, for example, from about 4 L/min toabout 6 L/min, e.g. from about 4.5 L/min to about 5.5 L/min, includingabout 5 L/min.

In one embodiment of the invention, the lipid and aminoglycoside (e.g.,amikacin) solutions are both filtered, for example through one or more(e.g., two in series) about 0.2 μm filters, prior to mixing into acombined stream (FIG. 1). Although FIG. 1 shows two filters in series,it should be noted that this number can be changed according to thepreference of the user of the method. For example, one to five filterscan be used to initially filter the lipid stream and the aminoglycosidestream.

In another embodiment, the aqueous saline solution (e.g., 1.5% salinesolution) is also filtered, for example through one or more (e.g., twoin series) about 0.2 μm filters, prior to mixing with thelipid-aminoglycoside combined stream in the reaction vessel. In afurther embodiment, the liposomal suspension, comprising liposomesformed at the intersection of the lipid and aminoglycoside streams,and/or in the combined lipid-aminoglycoside stream, is concentratedwithin the reaction vessel using a recirculating filtration system suchas diafiltration. As provided above, “encapsulation efficiency”, as usedherein, refers to the amount of aminoglycoside encapsulated or complexedwith liposomes prior to a filtration step, e.g., tangential flowfiltration of the liposomal aminoglycoside formulation to remove freeaminoglycoside. For example, an encapsulation efficiency of betweenabout 40% and about 70% (e.g., from about 45% to about 55%) can beachieved by mixing the lipid and aminoglycoside (e.g. amikacin) streamsaccording to the method of this invention as herein described.

In another embodiment, the resulting concentrated liposomal suspensionis treated (i.e., “washed”) with additional aqueous saline solution(e.g., filtered 1.5% saline solution) and subjected to furtherfiltration using a recirculating filtration system such as diafiltrationuntil the liposomal suspension contains an appropriate finalaminoglycoside concentration and substantially all of the freeaminoglycoside is removed. In a further embodiment, three or more washes(e.g., 3, 4, 5 or 6 washes) are conducted to achieve the appropriatefinal aminoglycoside concentration.

In one embodiment, following washing, the concentration ofaminoglycoside present in the liposomal aminoglycoside formulationmanufactured on a large-scale according to a method provided herein isabout 10 g/L or greater. In a further embodiment, aminoglycoside ispresent in the formulation at a concentration of about 20 g/L orgreater. In a further embodiment, aminoglycoside is present in theformulation at a concentration of about 30 g/L or greater. In a furtherembodiment, aminoglycoside is present in the formulation at aconcentration of about 40 g/L or greater. In a further embodiment,aminoglycoside is present in the formulation at a concentration of about50 g/L or greater. In a further embodiment, aminoglycoside is present inthe formulation at a concentration of about 60 g/L or greater. In afurther embodiment, aminoglycoside is present in the formulation at aconcentration of about 70 g/L or greater. In another embodiment, theaminoglycoside is present in the formulation at a concentration of fromabout 10 g/L to about 100 g/L. In a further embodiment, theaminoglycoside is amikacin. In one embodiment, the aminoglycoside ispresent in the formulation at a concentration of from about 50 g/L toabout 100 g/L. In a further embodiment, the aminoglycoside is amikacin.In one embodiment, the aminoglycoside is present in the formulation at aconcentration of from about 60 g/L to about 80 g/L. In a furtherembodiment, the aminoglycoside is amikacin. In yet another embodiment,the aminoglycoside is present in the formulation at a concentration fromabout 65 g/L to about 80 g/L. In a further embodiment, theaminoglycoside is amikacin. In yet another embodiment, theaminoglycoside is present in the formulation at a concentration fromabout 65 g/L to about 75 g/L. In a further embodiment, theaminoglycoside is amikacin. In another embodiment, amikacin is presentin the formulation at a concentration of about 70 g/L. In a furtherembodiment, the aminoglycoside is amikacin.

In a further embodiment, following washing, the concentration of lipidpresent in the liposomal drug formulation manufactured on a large-scaleaccording to a method provided herein is from about 10 g/L to about 100g/L, including about 20 g/L to about 80 g/L and about 40 g/L to about 60g/L (e.g., about 50 g/L).

In another embodiment, following diafiltration, thelipid-to-aminoglycoside weight ratio in a liposomal drug formulationmanufactured on a large-scale according to a method provided herein isless than 1:1, for example between about 0.5:1 (lipid:aminoglycoside)and about 0.8:1 (lipid:aminoglycoside) (e.g., about 0.5:1(lipid:aminoglycoside) or 0.6:1 (lipid:aminoglycoside) or 0.7:1(lipid:aminoglycoside) or 0.8:1 (lipid:aminoglycoside)). In oneembodiment, the lipid-to-aminoglycoside weight ratio is about 0.7:1(lipid:aminoglycoside).

The liposomal aminoglycoside formulation manufactured on a large-scaleaccording to a method provided herein comprises liposome particles witha mean particle size (i.e. a mean diameter) of from about 200 nm toabout 500 nm, for example from about 200 nm to about 400 nm (e.g. fromabout 250 nm to about 350 nm). The liposome diameter may be measuredusing commercially available light scattering technology, for example byquasi-elastic light scattering using a Nicomp™ 380 submicron particlesizer (Nicomp, Santa Barbara, Calif. USA).

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments and aspects described above, are illustrative andare not to be construed as limiting the scope of the invention in anyway.

EXAMPLES Example 1: Manufacturing Process and Process Controls forLiposomal Amikacin

The manufacture of liposomal amikacin sulfate was conducted using anaseptic process that involves the preparation of three sterile solutionstreams, mixing the lipid and amikacin sulfate streams at appropriateflow rates via a T-connector infusion module, collecting the combinedlipid-amikacin sulfate streams containing liposomes with encapsulatedamikacin sulfate in a sterilized diafiltration (reaction) vessel, addinga stream of 1.5% aqueous sodium chloride at an appropriate flow rate tothe diafiltration vessel, followed by diafiltration (including washing)and concentration of the resulting liposomal dispersion to form thefinal product.

a) Solution Preparation: Sufficient quantities of the following threesolutions were prepared.

-   -   Amikacin sulfate solution: Amikacin sulfate in water for        injection (WFI), pH adjusted with sodium hydroxide to 6.6-6.8.    -   Lipid solution: DPPC/cholesterol (2:1 w/w) in ethanol.    -   1.5% Sodium chloride solution: 1.5% Sodium chloride in WFI, pH        adjusted to 6.6-6.8.        The solutions must be used within 24 hours of preparation.        b) Infusion/Initial Concentration: The amikacin sulfate solution        and lipid solution were warmed and passed through separate        sterilizing filters before flowing through an in-line        T-connector infusion module at controlled rates of addition. The        mixed streams were collected in a pre-sterilized reactor vessel.        Simultaneously, 1.5% aqueous sodium chloride solution was passed        through a sterilizing filter and introduced as a stream at an        appropriate flow rate into the reactor vessel. At this stage,        the solution may be sampled for the level of amikacin        encapsulation.        c) Diafiltration: Diafiltration was conducted. This step        functions to remove the ethanol from the bulk solution and to        wash away any “unentrapped” or free amikacin sulfate.        d) Final Concentration: Using an in-process test result, the        bulk solution was concentrated to an appropriate concentration        level of amikacin sulfate. After concentration is complete,        confirmatory tests for concentration of amikacin sulfate and L/D        ratio may be performed.        Table 1 describes experiments (A) and (B), performed according        to the general method of Example 1. In (A), a ⅜″ T-connector        infusion module is used. In (B), a 3/16″ T-connector infusion        module is used.

TABLE 1 Amikacin Amikacin Amikacin Amikacin calculated sulfate sulfateLipid Lipid sulfate free base DPPC solution solution solution solutionL/D concentration weight weight Cholesterol concentration flow rateconcentration flow rate ratio obtained Ex. (kg) (kg) weight (kg) (g/L)(kg/min) (g/L) (kg/min) obtained (mg/mL) (A) 30.428 7.33 ± 3.67 ± 455.64 20 3.62 0.72 70 ± 3 0.050 0.050 (B) 8.250 1.666 ± 0.834 ± 45 1.46420 0.851 0.68 to 70 ± 3 0.001 0.001 0.74

In additional experiments, generally following the process of Example 1,the lipid and amikacin stream (flow) rates were varied, and theresulting concentrations of lipid and amikacin in the liposomalformulations were measured. The L/D ratio for each experiment wascalculated and the results presented in FIG. 2. The results provideguidance for an optimal relative lipid/amikacin flow rate to achieve apreferred L/D ratio.

All, documents, patents, patent applications, publications, productdescriptions, and protocols which are cited throughout this applicationare incorporated herein by reference in their entireties for allpurposes.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

1. A large-scale method of preparing a liposomal aminoglycosideformulation comprising a lipid and an aminoglycoside, wherein theoverall lipid-to-drug weight ratio is less than 1:1, the methodcomprising: (a) mixing a first stream comprising the lipid with a secondstream comprising the aminoglycoside to form a combinedlipid-aminoglycoside stream, (b) mixing the lipid-aminoglycoside streamof Step (a) with an aqueous saline solution in a reaction vessel, and(c) washing the product of Step (b) comprising the liposomalaminoglycoside formulation to remove unencapsulated aminoglycoside,wherein the relative flow rate ratio of the second stream to the firststream is about 1.5:1 to about 2:1.
 2. The method of claim 1, whereinthe second stream comprises an aqueous solution of amikacin.
 3. Themethod of claim 1, wherein the first stream comprises an alcoholicsolution of lipid.
 4. The method of claim 1, wherein the aqueous salinesolution is added to the reaction vessel via a third stream.
 5. Themethod of claim 4, wherein the third stream is added to the reactionvessel at the same time as the lipid-aminoglycoside stream.
 6. Themethod of claim 4, wherein the third stream is added to the reactionvessel prior to the lipid-aminoglycoside stream.
 7. The method of anyone of claims 1-6, wherein the flow rate of the first stream is fromabout 0.5 kg/min to about 1.5 kg/min, and the flow rate of the secondstream is from about 1 kg/min to about 2 kg/min.
 8. The method of anyone of claims 1-6, wherein the flow rate of the first stream is fromabout 3 kg/min to about 4 kg/min, and the flow rate of the second streamis from about 5 kg/min to about 7 kg/min.
 9. The method of claim 6,wherein the flow rate of the first stream is from about 0.5 kg/min toabout 1.5 kg/min, the flow rate of the second stream is from about 1kg/min to about 2 kg/min, and the flow rate of the third stream is fromabout 1 L/min to about 2 L/min.
 10. The method of claim 6, wherein theflow rate of the first stream is from about 3 kg/min to about 4 kg/min,the flow rate of the second stream is from about 5 kg/min to about 7kg/min, and the flow rate of the third stream is from about 3 L/min toabout 6 L/min.
 11. The method of any one of claims 1-10, wherein theaqueous saline solution is 1.5% sodium chloride.
 12. The method of anyone of claims 1-11, wherein the aminoglycoside is a pharmaceuticallyacceptable salt of the aminoglycoside.
 13. The method any one of claims1-12, wherein the lipid comprises a phospholipid.
 14. The method ofclaim 13, wherein the phospholipid is a phosphatidylcholine.
 15. Themethod of claim 14, wherein the phosphatidylcholine isdipalmitoylphosphatidylcholine (DPPC).
 16. The method of any one ofclaims 1-15, wherein the lipid comprises a sterol.
 17. The method ofclaim 16, wherein the sterol is cholesterol.
 18. The method of any oneof claims 1-17, wherein the lipid comprises DPPC and cholesterol. 19.The method of any one of claims 1-18, wherein the drug and lipid streamsare each maintained at a temperature from about 30° C. to about 50° C.prior to mixing.
 20. The method of claim 19, wherein the drug and lipidstreams are each maintained at a temperature from about 35° C. to about45° C. prior to mixing.
 21. The method of any one of claims 1-20,wherein the temperature of the combined lipid-aminoglycoside stream iscooled by the aqueous saline solution in the reaction vessel.
 22. Themethod of any one of claims 1-21, wherein following Step (b) liposomesare prepared with an aminoglycoside encapsulation efficiency of at least40%.
 23. The method of any one of claims 1-22, wherein liposomes areformed in the combined lipid-aminoglycoside stream.
 24. The method ofany one of claims 1-23, for the preparation of a liposomal drugformulation where the overall lipid-to-drug weight ratio is about 0.7:1.25. The method of any one of claims 1-23, wherein the washing Step (c)is performed using 1.5% aqueous sodium chloride solution.
 26. The methodof claim 25, wherein the washing Step (c) is repeated, and the productconcentrated to provide a liposomal drug formulation with theaminoglycoside present at a concentration of from about 60 g/L to about80 g/L.
 27. The method of any one of claims 1-26, wherein theaminoglycoside is arbekacin, astromicin, capreomycin, dibekacin,framycetin, gentamicin, hygromycin B, isepamicin, kanamycin, neomycin,netilmicin, paromomycin, rhodestreptomycin, ribostamycin, sisomicin,spectinomycin, streptomycin, tobramycin, verdamicin, or a combinationthereof.
 28. The method of any one of claims 1-26, wherein theaminoglycoside is AC4437, amikacin, apramycin, arbekacin, astromicin,bekanamycin, boholmycin, brulamycin, capreomycin, dibekacin, dactimicin,etimicin, framycetin, gentamicin, H107, hygromycin, hygromycin B,inosamycin, K-4619, isepamicin, KA-5685, kanamycin, neomycin,netilmicin, paromomycm, plazomicin, ribostamycin, sisomicm,rhodestreptomycin, sorbistin, spectinomycin, sporaricin, streptomycin,tobramycin, verdamicin, vertilmicin, or a combination thereof.
 29. Themethod of any one of claims 1-26, wherein the aminoglycoside isamikacin.
 30. The method of claim 29, wherein the amikacin is amikacinsulfate.
 31. A liposomal drug formulation manufactured by the method ofany one of claims 1-30.
 32. The liposomal drug formulation of claim 31,wherein the aminoglycoside is present at a concentration from about 60g/L to about 80 g/L.
 33. The liposomal drug formulation of claim 31,wherein amikacin is present at a concentration of about 70 g/L.
 34. Theliposomal drug formulation of any one of claims 31-33, wherein the lipidis present at a concentration from about 40 g/L to about 60 g/L.
 35. Theliposomal drug formulation of claim 34, wherein the lipid is present ata concentration of about 50 g/L.